OVERVIEW

Our research aims to understand how signals at the plasma membrane regulate the actin-based cytoskeleton, cell movement and proliferation. We are particularly interested in signal transduction mechanisms that are mediated through changes in intracellular pH, and how H+ fluxes at the plasma membrane drive physiological processes. H+ fluxes across biological membranes regulate cell proliferation, cell adhesion, cytoskeleton dynamics, vesicular trafficking, apoptosis, and bacterial and viral entry into mammalian cells. Our work focuses on H+ fluxes at the plasma membrane generated by ion exchangers, with an emphasis on the Na-H exchanger, NHE1.

CYTOSKELETON DYNAMICS and CELL MIGRATION

H+ efflux plays an evolutionarily conserved role in regulating cytoskeleton dynamics and cell migration, although through poorly understood mechanisms. We found that increased H+ efflux at the leading edge of migrating fibroblasts [Denker, J Cell Biol 159:1087; cited in Faculty of 1000] and chemotaxing Dictyostelium cells [Patel and Barber, J Cell Biol 169:321] is necessary for cell polarity, de-novo actin polymerization, and efficient cell movement (see quicktime files in website movie link). We also found that a leading-edge H+ efflux plays a central role in positive feedback loops for generating asymmetric amplification of signaling molecules during cell migration and chemotaxis, including production of PI(3,4,5)P3 and activation of the GTPases Rac and Cdc42 (Fig. 1). We are applying these findings to biological processes such as tumor cell metastasis, neuronal growth cone formation, and how cells sense and respond to mechanical forces. We also are determining at the molecular level how protons function as a post-translational mechanism to regulate protein activities, binding affinities, and stability (see Protonation paragraph below).

Our recent work on cytoskeleton dynamics and cell migration includes identifying a previously unrecognized mechanism for regulating the Arp2/3 complex. The Arp2/3 complex, an assembly of seven subunit proteins, drives an actin-based motility engine that builds crosslinked filament arrays to build membrane protrusions and phagocytic cups, and to drive cell migration, move endosomes, and engulf bacteria. Previous work identified members of the WASP/Scar-WAVE families of nucleation promoting factors as the sole mechanism for activating Arp2/3 and generating branched actin networks. However, we found that phosphorylation of the Arp2 subunit is necessary for activation of the Arp2/3 complex. We identified evolutionarily-conserved phosphorylation sites on Arp2, found that growth factors, oncogenes, and mechanical force increase Arp2 phosphorylation in normal and cancer cells, and generated computational models predicting how Arp2 phosphorylation induces activation of the Arp2/3 complex (Fig. 2). We currently are using mutational analyses to test our model predictions, library screens to identify kinases phosphorylating Arp2, and RNAi strategies to determine the role of regulatedArp2 phosphorylation in epithelial-mesenchymal transition, tumorigenesis, and metastasis.

PROTONATION AS A POSTTRANSLATIONAL SIGNALING MECHANISM

Changes in intracellular pH (pHi) regulate normal and pathological cell processes. Increases in pHi are necessary for growth factor-induced cell proliferation and differentiation, dynamic cytoskeletal remodeling and polarity during cell migration, neoplastic transformation and tumor progression, and viral and bacterial entry. Decreases in pHi promote vesicular trafficking and caspase-dependent and -independent apoptosis. Despite established effects of pHi on diverse cell functions, our understanding of intracellular pH sensors and the functional significance of their protonation state is limited. We recently began a very exciting collaborative project with Matt Jacobson, a computational biologist at UCSF, and Mark Kelly, an NMR spectroscopist at UCSF, to model and examine allosteric regulation of proteins by protons. Our diverse expertise facilitates a comprehensive analysis of the function, biochemistry, structure, and thermodynamics of intracellular pH sensors. Our initial focus is on sensitive pH sensors regulating actin dynamics, but our long-term objective is to identify design principles of such sensors. Understanding these principles will ultimately help to identify new pH-sensing proteins and, more speculatively, could lead to the rational engineering of pH-sensitive proteins.

We are studying two biological processes to test our hypotheses on H+-dependent regulation: (1) dynamic remodeling of cell-substrate adhesions and (2) actin polymerization, which are both dependent on increased pHi and necessary for cell motility. A key protein in adhesion remodeling is talin, which tethers the actin cytoskeleton to adhesion sites by an N-terminal binding to integrin adhesion molecules and an exquisitely pH-sensitive C-terminal binding to F-actin (Fig. 3A). A model constructed by the Jacobson lab reveals that 5 amino acids with pKa s near the physiological range cluster at one end of a 5-helix bundle (Fig. 3B). Constant-pH molecular dynamics simulations suggest a specific mechanism for how this pH sensor may modulate actin binding. At lower pH, increased protonation of the pH sensor causes the USH to pack less tightly against the other helices, potentially exposing a cryptic actin binding site in the I/LWEQ domain. The Barber lab tested and confirmed this model by showing that pH-dependent actin binding requires an interaction between the USH and I/LWEQ domains (Fig. 3C), and the Kelly lab is using NMR to determine pH-dependent solution structures of the I/LWEQ domain in the absence and presence of the USH segment. Based on modeling predictions, we generated mutant talins designed to be pH-insensitive, which we are testing biochemically for pH-dependent actin binding, and functionally for effects on focal adhesion remodeling and cell migration. Longer term, we are intrigued by other I/LWEQ domains in actin binding proteins, particularly Hip1, which is part of the clathrin complex necessary for endosome trafficking, and whether this domain represents a modular pH sensor.

We found that increased pHi is necessary for rapid formation of actin free barbed ends, an initial first-step in actin assembly that is regulated by a pH-dependent severing by cofilin. The Jacobson lab is modeling conformational changes in cofilin by protonation state of its His133 and by phosphorylation state of its Ser3, which both regulate cofilin severing activity. The Barber lab generated mutant pH-insensitive cofilins based on modeling predictions and is biochemically testing their actin binding and severing activity, and functionally testing their regulation of actin assembly and migration in mammalian fibroblasts and in Dictyostelium cells. The Kelly lab is using NMR to determine the pH-dependent structures of wild-type and mutant cofilins.

CELL PROLIFERATION

Increased cytosolic pH has a permissive effect in promoting cell proliferation. We are using genetic approaches and DNA arrays [Putney and Barber, BMS Genomics 16:46] to identify target genes that control the growth-promoting effects of increased cytosolic pH. Our recent findings indicate that at the end of S phase NHE1 generates a transient increase in intracellular pH that times G2/M entry and progression [Putney and Barber J Biol Chem 278:44645; cited in Faculty of 1000]. Blocking H+ efflux delays G2/M progression, inhibits the kinase activity of Cdc2 and the expression of cyclin B, and increases expression of the Cdc2 inhibitory kinase Wee 1. We currently are investigating the hypothesis that incrased H+ efflux at the end of S phase constitutes a previously unrecognized pH-dependent component of a G2/M checkpoint.

SELECTED RECENT PUBLICATIONS

Denker, S.P. Huang, D.C., Orlowski, J., Furthmayr, H., and Barber, D.L. 2000 Direct binding the Na-H exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton and cell shape independently of H+ translocation. Mol. Cell 6:1425-1436.

Denker, S.P., Yan, W.Y., and Barber, D.L. 2000 Effect of Rho GTPases on Na-H exchanger in mammalian cells. Methods Enzymol. 325:334-368.

Denker, S.P. Huang, D.C., Orlowski, J., Furthmayr, H., and Barber, D.L. 2000 Direct binding the Na-H exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton and cell shape independently of H+ translocation. Mol. Cell 6:1425-1436.

Yan, W., Nerke, K., J., Choi, J., and Barber, D.L. 2001 The Nck-interacting kinase (NIK) phosphorylates the Na-H exchanger NHE1 and regulates NHE1 activation by platelet-derived growth factor. J. Biol Chem. 276:31349-31356.

Putney, L., Denker, S.P., Barber, D.L. 2002 The changing face of the Na-H exchanger, NHE1: structure, regulation, and cellular actions. Annu. Rev. Pharmacol. Toxicol. 42:527-552. Denker, S.P., Barber, D.L. 2002 Ion transport proteins anchor and regulate the cytoskeleton. Curr. Opin. Cell Biol. 14:214-220.

Buchan, A.M.J., Lin, C.-Y., Choi, J., Barber, D.L. 2002 Somatostatin acting at receptor subtype SSTR1, inhibits Rho activity, the assembly of actin stress fibers, and cell migration. J. Biol Chem 277:28431-28438.

Denker, S.P. and Barber, D.L. 2002 Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1. J Cell Biol 159:1087-1096. (cited in Faculty of 1000 with 8.1 factor)

Lin, C-Y, Varma, MG, Joubel, A, Madabushi, S, Lichtarge, O and Barber, DL. 2003 Conserved motifs common to somatostatin, D2-dopamine, and alpha2B-adrenergic receptors for inhibiting the Na-H exchanger NHE1. J. Biol. Chem. 278:15128-15135.

Putney, L.K., and Barber, D.L. 2003. Na-H exchange-dependent increase in intracellular pH times G2/M entry and transition. J Biol Chem 278:44645-44649. (cited in Faculty of 1000 with 4.8 factor)

Putney, L.K., and Barber, D.L. 2004. Expression profile of genes regulated by activity of the Na-H exchanger NHE1. BMC Genomics 16:46-59.

Baumgartner, M., Patel, H., and Barber, DL. 2004 The Na-H exchanger NHE1 as a plasma membrane scaffold in the assembly of signaling complexes. Am. J. Physiol: Cell Physiol 287:C844-C805.

Patel, H. and Barber, DL. 2005 A developmentally-regulated Dictyostelium Na-H exchanger is necessary for cell polarity and chemotaxis J Cell Biol 169:321-329.

Baumgartner, M., Blackwood, E.M., Sillman, A., Srivastava, J., Madson, N., Schilling, J.W., Wright, J.H., and Barber, D.L. The Nck-interacting kinase NIK phosphorylates ERM proteins for formation of lamellipodium by growth factors. Proc. Natl. Acad. Sci. 2006 Sep 5;103(36):13391-6. Epub 2006 Aug 25.

Srivastava, J., Barber, D.L., and Jacobson, M.P. Intracellular pH sensors: design principles and functional significance. Physiology 2007 Feb;22:30-9.

Frantz, C., Karydis, A., Nalbant, P., Hahn K.M., Barber D.L. Positive feedback between Cdc42 activity and H+ efflux by the Na-H exchanger NHE1 for polarity of migrating cells. J Cell Biol., 2007 Nov 5;179(3):403-10.

LeClaire, LL III, Baumgartner, M, Iwasa, JH, Mullins, RD and Barber, DL. 2008 Phosphorylation of the Arp2/3 complex is necessary to nucleate actin filaments. J. Cell Biol. (in press).

Srivastava, J, Barreiro, G, Groscurth, S, Gringas, AR, Goult, BT Critchley, DR, Kelly, M.J.S, Jacobson. MP and Barber. DL. Structural model and functional significance of pH-dependent talin-actin binding for focal adhesion remodeling. Proc. Natl. Acad. Sci. (in press).